A general LC/MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures

ABSTRACTA complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and noncanonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these nonstandard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via integration of a hydrophobic end-labeling strategy with of 2-D mass-retention time LC/MS analysis to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA vs. its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site of the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification types and can also be used in the quality control of modified therapeutic RNAs.

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A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures

Nucleic Acids Research ◽

10.1093/nar/gkz731 ◽

2019 ◽

Vol 47 (20) ◽

pp. e125-e125 ◽

Cited By ~ 8

Author(s):

Ning Zhang ◽

Shundi Shi ◽

Tony Z Jia ◽

Ashley Ziegler ◽

Barney Yoo ◽

...

Keyword(s):

Rna Sequencing ◽

De Novo ◽

Direct Analysis ◽

Complete Sequence ◽

Complete Understanding ◽

Sequencing Method ◽

Single Base Resolution ◽

Base Modifications ◽

Real Complex ◽

Usage Efficiency

Abstract A complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and non-canonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these non-standard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via introduction of a 2-dimensional hydrophobic end-labeling strategy into traditional mass spectrometry-based sequencing (2D HELS MS Seq) to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA versus its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site in the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification type and can also be used in the quality control of modified therapeutic RNAs.

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Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

eLife ◽

10.7554/elife.27024 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 45

Author(s):

Jason C Bell ◽

David Jukam ◽

Nicole A Teran ◽

Viviana I Risca ◽

Owen K Smith ◽

...

Keyword(s):

Rna Sequencing ◽

Rna Processing ◽

De Novo ◽

Critical Component ◽

Proximity Ligation ◽

Ribonucleoprotein Complexes ◽

Genome Wide ◽

Sequencing Method ◽

Nascent Transcripts ◽

Drosophila Cells

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

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Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

10.1101/118786 ◽

2017 ◽

Author(s):

Jason C. Bell ◽

David Jukam ◽

Nicole A. Teran ◽

Viviana I. Risca ◽

Owen K. Smith ◽

...

Keyword(s):

Rna Sequencing ◽

Dosage Compensation ◽

Rna Processing ◽

De Novo ◽

Critical Component ◽

Proximity Ligation ◽

Ribonucleoprotein Complexes ◽

Genome Wide ◽

Sequencing Method ◽

Nascent Transcripts

AbstractRNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing.

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Quantitative profiling of N6-methyladenosine at single-base resolution in stem-differentiating xylem of Populus trichocarpa using Nanopore direct RNA sequencing

Genome Biology ◽

10.1186/s13059-020-02241-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yubang Gao ◽

Xuqing Liu ◽

Bizhi Wu ◽

Huihui Wang ◽

Feihu Xi ◽

...

Keyword(s):

Rna Sequencing ◽

Populus Trichocarpa ◽

Alternative Polyadenylation ◽

High Accuracy ◽

Single Base ◽

Rna Immunoprecipitation ◽

Single Base Resolution ◽

Single Transcript ◽

Quantitative Profiling ◽

Identification And Quantification

AbstractThere are no comprehensive methods to identify N6-methyladenosine (m6A) at single-base resolution for every single transcript, which is necessary for the estimation of m6A abundance. We develop a new pipeline called Nanom6A for the identification and quantification of m6A modification at single-base resolution using Nanopore direct RNA sequencing based on an XGBoost model. We validate our method using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and m6A-sensitive RNA-endoribonuclease–facilitated sequencing (m6A-REF-seq), confirming high accuracy. Using this method, we provide a transcriptome-wide quantification of m6A modification in stem-differentiating xylem and reveal that different alternative polyadenylation (APA) usage shows a different ratio of m6A.

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Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains

Plants ◽

10.3390/plants10040753 ◽

2021 ◽

Vol 10 (4) ◽

pp. 753

Author(s):

Miroslav Glasa ◽

Richard Hančinský ◽

Katarína Šoltys ◽

Lukáš Predajňa ◽

Jana Tomašechová ◽

...

Keyword(s):

High Throughput ◽

Potato Virus ◽

Mixed Infection ◽

High Throughput Sequencing ◽

Potato Virus Y ◽

De Novo ◽

Sweet Pepper ◽

Complete Sequence ◽

Genome Region ◽

Mapping Parameters

In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

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Construction of individual ddRAD libraries v2

10.17504/protocols.io.bv4tn8wn ◽

2021 ◽

Author(s):

Claire Daguin Thiebaut ◽

Stephanie Ruault ◽

Charlotte Roby ◽

Thomas Broquet ◽

Frédérique Viard ◽

...

Keyword(s):

Magnetic Beads ◽

Sex Chromosome ◽

De Novo ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Tree Frog ◽

Size Selection ◽

Hyla Arborea ◽

Sequencing Method ◽

Sample Representation

This protocol describes a double digested restriction-site associated DNA (ddRADseq) procedure, that is a variation on the original RAD sequencing method (Davey & Blaxter 2011), which is used for de novo SNP discovery and genotyping. This protocol differs from the original ddRADseq protocol (Peterson et al 2012), in which the samples are pooled just after the ligation to adaptors (i.e. before size selection and PCR). The present ddRAD protocol as been slightly adapted from Alan Brelsford's protocol published in the supplementary material of this paper: Brelsford, A., Dufresnes, C. & Perrin, N. 2016. High-density sex-specific linkage maps of a European tree frog (Hyla arborea) identify the sex chromosome without information on offspring sex. Heredity 116, 177–181 (2016). https://doi.org/10.1038/hdy.2015.83 In the present protocol, all samples are treated separately, in a microplate, until final PCR amplification performed before pooling. Despite being slightly more costly and time-consuming in the lab, it allows for fine adjustement of each sample representation in the final library pool, ensuring similar number of sequencing reads per sample in the final dataset. Briefly, genomic DNA from the samples are individually digested with 2 restriction enzymes (one rare-cutter and one more frequent cutter) then ligated to a barcoded adaptor (among 24 available) at one side, and a single adaptor at the other side, purified with magnetic beads, and PCR-amplified allowing the addition of a Illumina index (among 12 available) for multiplexing a maximum of 288 sample per library. Samples are then pooled in equimolar conditions after visualisation on an agarose gel. Purification and size selection is then performed before final quality control of the library and sequencing.

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Abstract 193: RNA-sequencing of Disease-specific iPSC as a New Filter to Identify Genes Associated With Hypoplastic Left Heart Syndrome

Circulation Research ◽

10.1161/res.117.suppl_1.193 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Almudena Martinez Fernandez ◽

Xing Li ◽

Jeanne L Theis ◽

Andre Terzic ◽

Timothy M Olson ◽

...

Keyword(s):

Rna Sequencing ◽

Hypoplastic Left Heart Syndrome ◽

De Novo ◽

Nuclear Family ◽

Cardiac Differentiation ◽

Left Heart ◽

Hypoplastic Left Heart ◽

Differentiated Cells ◽

Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is a complex multifactorial disease for which no definitive genetic causes have been found. Current genetic filtering strategies render lists of genes with unknown relevance in terms of pathogenesis. A complementary filter based on biological evidence would create a new approach to prioritize relevant candidate genes and mutations. In our study, 5 members of a nuclear family including a child with HLHS were evaluated using echocardiography and their genetic information was obtained through whole genome sequencing (WGS). Data filtering including rarity, functional impact and mode of inheritance was implemented, resulting in identification of 34 genes with recessive or de novo variants potentially involved in the pathogenesis of HLHS. Additionally, iPSC were derived from proband and parents and subjected to RNA-sequencing at the undifferentiated state and following spontaneous differentiation. Comparative transcriptional analyses identified genes differentially expressed in proband samples at each stage. These gene sets were used as an additional filter for the previously generated WGS data. This strategy revealed that out of 34 mutated genes originally identified, 10 displayed transcriptional differences in undifferentiated iPSC from the HLHS-affected individual while 16 out of 34 mutated genes showed significantly different expression levels in differentiated cells from proband. Furthermore, expression dynamics were studied during guided cardiac differentiation for the 9 genes fulfilling all applied criteria. Two genes not previously linked to HLHS, ELF4 and HSPG2 were found to behave significantly different in HLHS-iPSC when compared to control counterparts. In summary, filtering WGS data according to a new layer of transcriptional information that leverages iPSC plasticity allows prioritization of genes associated with HLHS in an in vitro model of disease.

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